In vitro plantlet regeneration from mature zygotic embryos of Pinus pinea L.: overcoming the rooting problems

Na natureza, as plantas e os microrganismos estabelecem associações de várias ordens. Nas culturas in vitro de plantas, mesmo as associações favoráveis com microrganismos foram, durante muitos anos, consideradas como contaminantes. Só mais tarde, as vantagens da inoculação in vitro (co-cultura) foram demonstradas e as técnicas de biotização (bacterização ou micorrização) usadas com o objetivo de melhorar as condições de crescimento in vitro. As dificuldades do enraizamento in vitro de uma das espécies mais importantes da floresta mediterrânica portuguesa, Pinus pinea L., conduziu à escolha deste sistema biológico, como matéria de estudo para a tese. Neste estudo, foram utilizados fungos ectomicorrízicos para otimizar a fase de enraizamento de plantas de Pinus pinea L. micropropagadas via organogénese. A introdução de ectomicorrízas no processo de micropropagação reativou o crescimento das raízes e induziu a melhoria dos vários parâmetros do sistema radicular adventício conduzindo a uma menor perda de plantas durante a aclimatização. Com efeito, a micorrização melhorou a funcionalidade das raízes, facilitando a absorção de nutrientes e de água. Neste trabalho, efetuou-se também uma extensiva caracterização morfológica e molecular das ectomicorrízas associadas a P. pinea. Das várias co-culturas testadas, selecionou-se a interação Pisolithus arhizus/P. pinea para estudar os sinais bioquímicos pré-simbióticos estabelecidos durante as etapas iniciais da co-cultura in vitro. Para possibilitar este estudo foi desenvolvido um novo sistema de co-cultura, o qual já está patenteado. Os resultados indicam que a presença de compostos fenólicos, nomeadamente o ácido o-coumarico, poderão ser importantes mediadores na interação fungo/planta.In nature, plants and microorganisms establish symbiotic associations of various orders. However, for many years such associations were deemed unnecessary in in vitro cultures because the culture medium provides ample amounts of nutrients and plant growth regulators to a growing plant. Only recently, the benefits of biotization (bacterization or mycorrhization) of plants regenerated in vitro were demonstrated by improvements in their growth and vigor. Pinus pinea L., which is of one of the most important species of Portuguese Mediterranean forests, can be regenerated in vitro from embryo cotyledons but the growth of adventitious roots induced in shoots ceases shortly after their formation. Overcoming this particular biological impediment was the study subject of the thesis. In this study, ectomycorrhizal fungi were used to improve adventitious rooting of Pinus pinea L. plants micropropagated through organogenesis. The introduction of ectomycorrhizae during the micropropagation process reactivated the root growth and improved several root characteristics leading to a reduced loss of plants during acclimatization. In fact, the mycorrhization enhanced root functionality facilitating the absorption of nutrients and water. In this work, an extensive characterization of morphological and molecular ectomycorrhizae associated with P. pinea was also undertaken. Of the several fungus species tested, the interaction of Pisolithus arhizus/P. pinea was selected for studying the pre-symbiotic biochemical signals established during the initial stages of co-culture in vitro. To facilitate this study, a novel co-culture system was developed which has been patented. The results indicate that the phenolic compounds, in particular the o coumaric acid ester might be important mediators in the interaction between the fungus and stone pine

Histological Studies of Mycorrhized Roots and Mycorrhizal-Like-Structures in Pine Roots

Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots

In vitro plantlet regeneration from mature zygotic embryos of Pinus pinea L.: overcoming the rooting problems

Regeneração in vitro de plântulas de embriões zigóticos maduros de Pinus pinea L.: superando os problemas de enraizamento Na natureza, as plantas e os microrganismos estabelecem associações de várias ordens. Nas culturas in vitro de plantas, mesmo as associações favoráveis com microrganismos foram, durante muitos anos, consideradas como contaminantes. Só mais tarde, as vantagens da inoculação in vitro (co-cultura) foram demonstradas e as técnicas de biotização (bacterização ou micorrização) usadas com o objetivo de melhorar as condições de crescimento in vitro. As dificuldades do enraizamento in vitro de uma das espécies mais importantes da floresta mediterrânica portuguesa, Pinus pinea L., conduziu à escolha deste sistema biológico, como matéria de estudo para a tese. Neste estudo, foram utilizados fungos ectomicorrízicos para otimizar a fase de enraizamento de plantas de Pinus pinea L. micropropagadas via organogénese. A introdução de ectomicorrízas no processo de micropropagação reativou o crescimento das raízes e induziu a melhoria dos vários parâmetros do sistema radicular adventício conduzindo a uma menor perda de plantas durante a aclimatização. Com efeito, a micorrização melhorou a funcionalidade das raízes, facilitando a absorção de nutrientes e de água. Neste trabalho, efetuou-se também uma extensiva caracterização morfológica e molecular das ectomicorrízas associadas a P. pinea. Das várias co-culturas testadas, selecionou-se a interação Pisolithus arhizus/P. pinea para estudar os sinais bioquímicos pré-simbióticos estabelecidos durante as etapas iniciais da co-cultura in vitro. Para possibilitar este estudo foi desenvolvido um novo sistema de co-cultura, o qual já está patenteado. Os resultados indicam que a presença de compostos fenólicos, nomeadamente o ácido o-coumarico, poderão ser importantes mediadores na interação fungo/planta; ABSTRACT: In nature, plants and microorganisms establish symbiotic associations of various orders. However, for many years such associations were deemed unnecessary in in vitro cultures because the culture medium provides ample amounts of nutrients and plant growth regulators to a growing plant. Only recently, the benefits of biotization (bacterization or mycorrhization) of plants regenerated in vitro were demonstrated by improvements in their growth and vigor. Pinus pinea L., which is of one of the most important species of Portuguese Mediterranean forests, can be regenerated in vitro from embryo cotyledons but the growth of adventitious roots induced in shoots ceases shortly after their formation. Overcoming this particular biological impediment was the study subject of the thesis. In this study, ectomycorrhizal fungi were used to improve adventitious rooting of Pinus pinea L. plants micropropagated through organogenesis. The introduction of ectomycorrhizae during the micropropagation process reactivated the root growth and improved several root characteristics leading to a reduced loss of plants during acclimatization. In fact, the mycorrhization enhanced root functionality facilitating the absorption of nutrients and water. In this work, an extensive characterization of morphological and molecular ectomycorrhizae associated with P. pinea was also undertaken. Of the several fungus species tested, the interaction of Pisolithus arhizus/P. pinea was selected for studying the pre-symbiotic biochemical signals established during the initial stages of co-culture in vitro. To facilitate this study, a novel co-culture system was developed which has been patented. The results indicate that the phenolic compounds, in particular the ocoumaric acid ester might be important mediators in the interaction between the fungus and stone pine

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved e.g. through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots including physical and chemical ones such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates or media (reviewed by Ragonezi et al. 2010). The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant- growth-promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests

Reference Genes Selection and Normalization of Oxidative Stress Responsive Genes upon Different Temperature Stress Conditions in Hypericum perforatum L

Abstract Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John’s wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes

Different seaweeds use for iodine deficiency overcome

Iodine deficiency is one of the three most common nutritional deficiencies worldwide and is a global public health problem that affects particularly the young children, pregnant women and elderly. The consequences of iodine deficiency disorder (IDD) include goiter and cretinism. The traditional consumption of iodine via supplemented salt should be monitored to reduce population sodium intake due to hypertension problems and the uncertain bioavailability of iodine in the supplemented salt. One approach for an effective and cost-efficient iodine supplementation is the use of seaweeds in food intake. Seaweeds, among all foods, are the most famous and reliable source of natural iodine. In this minireview we pretend to explore the different uses of seaweeds to fill the iodine deficiency and improve life quality.This work was supported by the projects Blue Iodine II “Boost Blue economy through market uptake an innovative seaweed bioextract for iodine fortification II” (733552, H2020- SMEInst-2016-2017); DemoBlueAlgae “Desenvolvimento de metodologias e optimização dos processos de cultivo e processamento de macroalgas para a industria e economia azul” PROCiência 2020 (M1420-01-0247-FEDER000002) and Madeira PO 14-20, contract CASBio, refª. M1420-01-0145-FEDER-000011.info:eu-repo/semantics/publishedVersio

Influence of light quality and intensity on adventitious root formation in microshoots of Pinus pinea L.

In the present study of Pinus pinea L., further improvement of microshoot rooting was achieved by applying Cool-white light at increased intensity from 60 to 90 µmol m-2 s-1. In contrast, light provided by Gro-lux lamps promoted rooting of the microshoots at the same frequency regardless of its intensity. Majority of microshoots (70.4%) grown under Cool-white lamps at the intensity of 90 µmol m-2 s-1 were also significantly taller when compared with those from other tested treatments

Can functional hologenomics aid tackling current challenges in plant breeding?

Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review in tends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping tools could greatly increase the efficiency of breeding programs.info:eu-repo/semantics/publishedVersio

Adventitious rooting of conifers: influence of physical and chemical factors

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species

Viral diagnosis in cultivars of Ipomoea batatas (L.) Lam

Ipomoea batatas (L.) Lam. commonly known as sweet potato, is an important staple food worldwide, mainly due to its high nutritional value and yield. However, vegetative reproduction of sweet potato makes it more susceptible to viral infections, which threatens its productivity, quality, and difficult long-term preservation in germplasm banks. Also, it can act as a virus reservoir infecting the rest of the plant accessions in the bank collections. Hence, this work aimed to screen Begomovirus, Potyvirus, and Carlavirus infections in 16 traditional sweet potato cultivars from the germplasm collection of the ISOPlexis Germplasm Bank, Madeira, Portugal. The infection prevalence by these viruses among cultivars was 81.25%, 25.00%, and 6.25%, respectively; being ISOP1011 the only accession coinfected by Potyvirus and Carlavirus. The accessions ISOP1006, ISOP1010, and ISOP1047 were also coinfected by Begomovirus and Potyvirus, highlighting their vulnerability to viral infections. The ISOP1005 and ISOP1027 accessions were the only ones not infected by any of these viruses. The analysis of the partial sequence obtained from the Carlavirus detected in the accession ISOP1011, revealed the existence of an ORF that encodes for 93 amino acids of the catalytic domain of an RNA-directed RNA polymerase related to the Tymovirus protein family, as could be confirmed by comparison with proteins stored in UniProtKB. Multiple sequence alignment with these proteins showed that Motifs A and B of the catalytic domain were conserved. The search for sequence similarity with sequences deposited in GenBank reported a high sequence identity with Sweet potato yellow mottle virus (SPYMV) and Sweet potato chlorotic fleck virus (SPCFV). However, the 9-11% discrepancy in nucleotide sequence identity and a phylogenetic analysis carried out using the maximum probability method suggests the virus isolated from ISOP1011 is a new divergent strain of the SPCFV species.info:eu-repo/semantics/publishedVersio